Systems genetics uncover new loci containing functional gene candidates in Mycobacterium tuberculosis-infected Diversity Outbred mice.

Mycobacterium tuberculosis infects two billion people across the globe, and results in 8-9 million new tuberculosis (TB) cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. Here, we investigate the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected with M. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using immune and inflammatory mediators; and clinical, microbiological, and granuloma correlates of disease identified five new loci on mouse chromosomes 1, 2, 4, 16; and three known loci on chromosomes 3 and 17. Further, multiple positively correlated traits shared loci on chromosomes 1, 16, and 17 and had similar patterns of allele effects, suggesting these loci contain critical genetic regulators of inflammatory responses to M. tuberculosis. To narrow the list of candidate genes, we used a machine learning strategy that integrated gene expression signatures from lungs of M. tuberculosis-infected Diversity Outbred mice with gene interaction networks to generate scores representing functional relationships. The scores were used to rank candidates for each mapped trait, resulting in 11 candidate genes: Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa, and Zfp318. Although all candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling, and all contain single nucleotide polymorphisms (SNPs), SNPs in only four genes (S100a8, Itgb5, Fstl1, Zfp318) are predicted to have deleterious effects on protein functions. We performed methodological and candidate validations to (i) assess biological relevance of predicted allele effects by showing that Diversity Outbred mice carrying PWK/PhJ alleles at the H-2 locus on chromosome 17 QTL have shorter survival; (ii) confirm accuracy of predicted allele effects by quantifying S100A8 protein in inbred founder strains; and (iii) infection of C57BL/6 mice deficient for the S100a8 gene. Overall, this body of work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and functionally relevant gene candidates that may be major regulators of complex host-pathogens interactions contributing to granuloma necrosis and acute inflammation in pulmonary TB.

History of tuberculosis disease is associated with genetic regulatory variation in Peruvians.

A quarter of humanity is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n = 63) or did not progress to TB (controls, n = 63). Transcriptomic profiling of monocyte-derived DCs and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Four genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

Intracellular Pathogens: Infection, Immunity, and Intervention.

Intracellular pathogens comprise a diverse group of pathogens that all share a required location in a host cell to infect, survive, and replicate. Intracellular location allows pathogens to hide from host immune responses, avoid competition with other pathogens, mediate host cellular functions, replicate safely, and cause infection that is difficult to target with therapeutics. All intracellular pathogens have varying routes of infiltration into host cells and different host cell preferences. For example, bacteria Mycobacterium tuberculosis chooses to invade antigen-presenting cells, which allows them to moderate host antigen presentation to memory cells, whereas rabies virus prefers to invade neurons because they have pre-existing innate immunity protection systems. Regardless of the pathway that each intracellular pathogen follows, all share the capacity to cause disease if they succeed in entering host cells. Here, we give an overview of selected intracellular pathogens and infections they cause, immune responses they induce, and intervention strategies used to treat and control them.

Host Cell Death and Modulation of Immune Response against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a prevalent infectious disease affecting populations worldwide. A classic trait of TB pathology is the formation of granulomas, which wall off the pathogen, via the innate and adaptive immune systems. Some key players involved include tumor necrosis factor-alpha (TNF-α), foamy macrophages, type I interferons (IFNs), and reactive oxygen species, which may also show overlap with cell death pathways. Additionally, host cell death is a primary method for combating and controlling Mtb within the body, a process which is influenced by both host and bacterial factors. These cell death modalities have distinct molecular mechanisms and pathways. Programmed cell death (PCD), encompassing apoptosis and autophagy, typically confers a protective response against Mtb by containing the bacteria within dead macrophages, facilitating their phagocytosis by uninfected or neighboring cells, whereas necrotic cell death benefits the pathogen, leading to the release of bacteria extracellularly. Apoptosis is triggered via intrinsic and extrinsic caspase-dependent pathways as well as caspase-independent pathways. Necrosis is induced via various pathways, including necroptosis, pyroptosis, and ferroptosis. Given the pivotal role of host cell death pathways in host defense against Mtb, therapeutic agents targeting cell death signaling have been investigated for TB treatment. This review provides an overview of the diverse mechanisms underlying Mtb-induced host cell death, examining their implications for host immunity. Furthermore, it discusses the potential of targeting host cell death pathways as therapeutic and preventive strategies against Mtb infection.

Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy.

We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.

Comparative analysis of genomic characteristics and immune response between Mycobacterium tuberculosis strains cultured continuously for 25 years and H37Rv.

Tuberculosis (TB) continues to pose a significant global health challenge, emphasizing the critical need for effective preventive measures. Although many studies have tried to develop new attenuated vaccines, there is no effective TB vaccine. In this study, we report a novel attenuated Mycobacterium tuberculosis (M. tb) strain, CHVAC-25, cultured continuously for 25 years in the laboratory. CHVAC-25 exhibited significantly reduced virulence compared to both the virulent H37Rv strain in C57BL/6J and severe combined immunodeficiency disease mice. The comparative genomic analysis identified 93 potential absent genomic segments and 65 single nucleotide polymorphic sites across 47 coding genes. Notably, the deletion mutation of ppsC (Rv2933) involved in phthiocerol dimycocerosate synthesis likely contributes to CHVAC-25 virulence attenuation. Furthermore, the comparative analysis of immune responses between H37Rv- and CHVAC-25-infected macrophages showed that CHVAC-25 triggered a robust upregulation of 173 genes, particularly cytokines crucial for combating M. tb infection. Additionally, the survival of CHVAC-25 was significantly reduced compared to H37Rv in macrophages. These findings reiterate the possibility of obtaining attenuated M. tb strains through prolonged laboratory cultivation, echoing the initial conception of H37Ra nearly a century ago. Additionally, the similarity of CHVAC-25 to genotypes associated with attenuated M. tb vaccine positions it as a promising candidate for TB vaccine development.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

Mycobacterium tuberculosis suppresses host antimicrobial peptides by dehydrogenating L-alanine.

Antimicrobial peptides (AMPs), ancient scavengers of bacteria, are very poorly induced in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis), but the underlying mechanism remains unknown. Here, we report that L-alanine interacts with PRSS1 and unfreezes the inhibitory effect of PRSS1 on the activation of NF-κB pathway to induce the expression of AMPs, but mycobacterial alanine dehydrogenase (Ald) Rv2780 hydrolyzes L-alanine and reduces the level of L-alanine in macrophages, thereby suppressing the expression of AMPs to facilitate survival of mycobacteria. Mechanistically, PRSS1 associates with TAK1 and disruptes the formation of TAK1/TAB1 complex to inhibit TAK1-mediated activation of NF-κB pathway, but interaction of L-alanine with PRSS1, disables PRSS1-mediated impairment on TAK1/TAB1 complex formation, thereby triggering the activation of NF-κB pathway to induce expression of AMPs. Moreover, deletion of antimicrobial peptide gene ß-defensin 4 (Defb4) impairs the virulence by Rv2780 during infection in mice. Both L-alanine and the Rv2780 inhibitor, GWP-042, exhibits excellent inhibitory activity against M. tuberculosis infection in vivo. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses its own alanine dehydrogenase to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

Fractal fractional model for tuberculosis: existence and numerical solutions.

This paper deals with the mathematical analysis of Tuberculosis by using fractal fractional operator. Mycobacterium TB is the bacteria that causes tuberculosis. This airborne illness mostly impacts the lungs but may extend to other body organs. When the infected individual coughs, sneezes or speaks, the bacterium gets released into the air and travels from one person to another. Five classes have been formulated to study the dynamics of this disease: susceptible class, infected of DS, infected of MDR, isolated class, and recovered class. To study the suggested fractal fractional model's wellposedness associated with existence results, and boundedness of solutions. Further, the invariant region of the considered model, positive solutions, equilibrium point, and reproduction number. One would typically employ a fractional calculus approach to obtain numerical solutions for the fractional order Tuberculosis model using the Adams-Bashforth-Moulton method. The fractional order derivatives in the model can be approximated using appropriate numerical schemes designed for fractional order differential equations.

Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Transcriptional activation of the Mycobacterium tuberculosis virulence-associated small RNA MTS1338 by the response regulators DosR and PhoP.

MTS1338, a distinctive small RNA in pathogenic mycobacteria, plays a crucial role in host-pathogen interactions during infection. Mycobacterial cells encounter heterogeneous stresses in macrophages, which highly upregulate MTS1338. A dormancy regulatory factor DosR regulates the intracellular abundance of MTS1338. Herein, we investigated the interplay of DosR and a low pH-inducible gene regulator PhoP binding to the MTS1338 promoter. We identified that DosR strongly binds to two regions upstream of the MTS1338 gene. The proximal region possesses a threefold higher affinity than the distal site, but the presence of both regions increased the affinity for DosR by > 10-fold. PhoP did not bind to the MTS1338 gene but binds to the DosR-bound MTS1338 gene, suggesting a concerted mechanism for MTS1338 expression.

The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host.

Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.

HSP-27 and HSP-70 negatively regulate protective defence responses from macrophages during mycobacterial infection.

Mycobacterium tuberculosis attenuates many defence responses from alveolar macrophages to create a niche at sites of infection in the human lung. Levels of Heat Shock Proteins have been reported to increase many folds in the serum of active TB patients than in latently infected individuals. Here we investigated the regulation of key defence responses by HSPs during mycobacterial infection. We show that infection of macrophages with M. bovis BCG induces higher expression of HSP-27 and HSP-70. Inhibiting HSP-27 and HSP-70 prior to mycobacterial infection leads to a significant decrease in mycobacterial growth inside macrophages. Further, inhibiting HSPs resulted in a significant increase in intracellular oxidative burst levels. This was accompanied by an increase in the levels of T cell activation molecules CD40 and IL-12 receptor and a concomitant decrease in the levels of T cell inhibitory molecules PD-L1 and IL-10 receptor. Furthermore, inhibiting HSPs significantly increased the expression of key proteins in the autophagy pathway along with increased activation of pro-inflammatory promoting transcription factors NF-κB and p-CREB. Interestingly, we also show that both HSP-27 and HSP-70 are associated with anti-apoptotic proteins Bcl-2 and Beclin-1. These results point towards a suppressive role for host HSP-27 and HSP-70 during mycobacterial infection.

Oxidation of the Mycobacterium tuberculosis key virulence factor protein tyrosine phosphatase A (MptpA) reduces its phosphatase activity.

The Mycobacterium tuberculosis tyrosine-specific phosphatase MptpA and its cognate kinase PtkA are prospective targets for anti-tuberculosis drugs as they interact with the host defense response within the macrophages. Although both are structurally well-characterized, the functional mechanism regulating their activity remains poorly understood. Here, we investigate the effect of post-translational oxidation in regulating the function of MptpA. Treatment of MptpA with H2 O2 /NaHCO3 , mimicking cellular oxidative stress conditions, leads to oxidation of the catalytic cysteine (C11) and to a conformational rearrangement of the phosphorylation loop (D-loop) by repositioning the conserved tyrosine 128 (Y128) and generating a temporarily inactive preclosed state of the phosphatase. Thus, the catalytic cysteine in the P-loop acts as a redox switch and regulates the phosphatase activity of MptpA.

The C terminus of the mycobacterium ESX-1 secretion system substrate ESAT-6 is required for phagosomal membrane damage and virulence.

SignificanceTuberculosis (TB), an ancient disease of humanity, continues to be a major cause of worldwide death. The causative agent of TB, Mycobacterium tuberculosis, and its close pathogenic relative Mycobacterium marinum, initially infect, evade, and exploit macrophages, a major host defense against invading pathogens. Within macrophages, mycobacteria reside within host membrane-bound compartments called phagosomes. Mycobacterium-induced damage of the phagosomal membranes is integral to pathogenesis, and this activity has been attributed to the specialized mycobacterial secretion system ESX-1, and particularly to ESAT-6, its major secreted protein. Here, we show that the integrity of the unstructured ESAT-6 C terminus is required for macrophage phagosomal damage, granuloma formation, and virulence.

Itaconate, Arginine, and Gamma-Aminobutyric Acid: A Host Metabolite Triad Protective Against Mycobacterial Infection.

Immune metabolic regulation shapes the host-pathogen interaction during infection with Mycobacterium tuberculosis (Mtb), the pathogen of human tuberculosis (TB). Several immunometabolites generated by metabolic remodeling in macrophages are implicated in innate immune protection against Mtb infection by fine-tuning defensive pathways. Itaconate, produced by the mitochondrial enzyme immunoresponsive gene 1 (IRG1), has antimicrobial and anti-inflammatory effects, restricting intracellular mycobacterial growth. L-arginine, a component of the urea cycle, is critical for the synthesis of nitric oxide (NO) and is implicated in M1-mediated antimycobacterial responses in myeloid cells. L-citrulline, a by-product of NO production, contributes to host defense and generates L-arginine in myeloid cells. In arginase 1-expressing cells, L-arginine can be converted into ornithine, a polyamine precursor that enhances autophagy and antimicrobial protection against Mtb in Kupffer cells. Gamma-aminobutyric acid (GABA), a metabolite and neurotransmitter, activate autophagy to induce antimycobacterial host defenses. This review discusses the recent updates of the functions of the three metabolites in host protection against mycobacterial infection. Understanding the mechanisms by which these metabolites promote host defense will facilitate the development of novel host-directed therapeutics against Mtb and drug-resistant bacteria.

Impact of pathobiological diversity of Mycobacterium tuberculosis on clinical features and lethal outcome of tuberculosis.

BACKGROUND: Mycobacterium tuberculosis population in Russia is dominated by the notorious Beijing genotype whose major variants are characterized by contrasting resistance and virulence properties. Here we studied how these strain features could impact the progression of pulmonary tuberculosis (TB) concerning clinical manifestation and lethal outcome. RESULTS: The study sample included 548 M. tuberculosis isolates from 548 patients with newly diagnosed pulmonary TB in Omsk, West Siberia, Russia. Strains were subjected to drug susceptibility testing and genotyping to detect lineages, sublineages, and subtypes (within Beijing genotype). The Beijing genotype was detected in 370 (67.5%) of the studied strains. The strongest association with multidrug resistance (MDR) was found for epidemic cluster Beijing B0/W148 (modern sublineage) and two recently discovered MDR clusters 1071-32 and 14717-15 of the ancient Beijing sublineage. The group of patients infected with hypervirulent and highly lethal (in a mouse model) Beijing 14717-15 showed the highest rate of lethal outcome (58.3%) compared to Beijing B0/W148 (31.4%; P = 0.06), Beijing Central Asian/Russian (29.7%, P = 0.037), and non-Beijing (15.2%, P = 0.001). The 14717-15 cluster mostly included isolates from patients with infiltrative but not with fibrous-cavernous and disseminated TB. In contrast, a group infected with low virulent 1071-32-cluster had the highest rate of fibrous-cavernous TB, possibly reflecting the capacity of these strains for prolonged survival and chronicity of the TB process. CONCLUSIONS: The group of patients infected with hypervirulent and highly lethal in murine model 14717-15 cluster had the highest proportion of the lethal outcome (58.3%) compared to the groups infected with Beijing B0/W148 (31.4%) and non-Beijing (15.2%) isolates. This study carried out in the TB high-burden area highlights that not only drug resistance but also strain virulence should be considered in the implementation of personalized TB treatment.

Mycobacterial infection aggravates Helicobacter pylori-induced gastric preneoplastic pathology by redirection of de novo induced Treg cells.

The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions.